The present invention relates to a highly active bioactivating substance having a high silicon component content. The bioactivating substance may be obtained by adding a silicate to an extracted substance from tissues activated by adding internal or external stressors to animals or animal tissues, or by carrying out a special extraction to make the content of silicic acid high in the extracted substance from the activated tissues.
Living organisms survive as an individual by adjusting and maintaining their physical and chemical states to and within certain stable physiological conditions by adapting to the changes in internal and external circumstances. To maintain such homeostasis, the living organism always produces various substances in vivo. Upon invasion by viruses or bacteria, and upon generation of tumor cells, it also produces resistant substances in vivo to such external and internal invasions.
However, when the biofunctional balance is disturbed and the unbalanced state becomes chronic, various diseases result. The ideal way of curing the disease is to recover from the abnormal state of the unbalanced biofunction to a normal state by invigorating and regulating homeostasis of the living body. It is well known that various receptors and ion channels such as sodium, potassium and calcium channels on cell surfaces especially carry out the maintenance and normalization of biofinctions. It is also known that, upon growing older, the ability of DNA to recover against damage decreases in mammalian cells and that the production of free radicals in vivo promotes aging and the generation of collagen disease and cancer. Collagen is a noncellular substance widely present in skin, blood vessels, cartilage, eyeballs and kidneys. A crosslinking of collageneous materials proceeds with aging and their elasticity decreases and they become hard.
Histamine is released from mast cells upon the antigen-antibody reaction and causes various allergic reactions. A suppressive ability for mast cell degranulation may contribute to normalization of the abnormal diseased state.
The present inventor has focused on the homeostatic functions of a living body, which regulate and restore the neurological, immunological and endocrinological disturbances due to functional abnormality in the diseased state. He has conducted an extensive investigation to ascertain resistant substances produced in vivo to external and internal stresses. As a result of such investigations on substances produced from activating living tissues, which enhance the natural curing activity and normalize the functions of a living body, the present inventor has found a highly active bioactivating substance and accomplished the present invention.
In the present invention, a high silicon component content, bioactivating substance is obtained from an extract from activated or stressed tissues and a silicate. The bioactivating substance of the invention is a biofunction-regulating and maintaining substance which may cure and normalize the abnormal functions occurring in a diseased state. It exhibits physiological activities such as unexpectedly superior suppressing action on histamine liberation and inhibition of hyaluronidase activity. It may be employed in pharmaceutically effective amounts in pharmaceutical compositions for the treatment of allergies in patients known to be in need of such treatment.
The present invention provides a highly active bioactivating substance comprising an extract from activated tissue. The bioactivating substance has a silicon component content of greater than 20 xcexcg, for example greater than 22 xcexcg, preferably greater than 25 xcexcg, which is calculated as silicon per mg of dried substance. To obtain the bioactivating substance of the present invention, various animals or animal tissues are inoculated with viruses or tumor cells as a stressor to activate the tissues, a physiologically active substance is extracted from the activated tissue and a soluble silicate is added thereto, The bioactivating substance of the present invention may also be prepared by a special extracting method to obtain a high content of silicic acid in the extracted substance from the activated tissue. In embodiments of the present invention, the ratio of the silicon component content of the extract from the activated tissue calculated as silicon to the total silicon component content of the bioactivating substance calculated as silicon is 1:3 to 1:10. The bioactivating substance of this invention exhibits physiological activities such as unexpectedly high suppressing action on histamine liberation and inhibition of hyaluronidase activity. The bioactivating substance of the invention is a biofunction-regulating and maintaining substance which may cure and normalize abnormal functions occurring in a diseased state.
The present invention provides a bioactivating substance which suppresses histamine liberation from mast cells and inhibits hyaluronidase activity comprising an extract from activated tissue. The extract and the bioactivating substance containing it comprise at least one silicon component. In embodiments of the invention, at least one additional silicon component is admixed with the extract from activated tissue to obtain a mixture, and the mixture is dried to obtain a powder form of the bioactivating substance of the present invention. The bioactivating substance of the present invention, and the dried extract from the activated tissues, exhibit positive color reactions to amino acid (by a ninhydrin reaction), saccharide (by an orcinol-iron (III) chloride-hydrochloric acid method), phosphorus (by a molybdenum blue method) and silicic acid (by a molybdenum blue method), and negative qualitative reactions to protein (by a trichloroacetic acid method) and phenol (by a ferric chloride method). The bioactivating substance of the present invention obtained by admixing the extract from the activated tissue and the additional silicon component, has a silicon component content which is more than 20 xcexcg calculated as silicon per mg of dried substance. The additional silicon component substantially increases the silicon component content of the extract from activated tissues and substantially increases its physiological activity. In embodiments of the present invention, the bioactivating substance may have a silicon component content of greater than 22 xcexcg, preferably greater than 25 xcexcg, which is calculated as silicon per mg of dried substance. The ratio of the silicon component content of the extract from the activated tissue calculated as silicon to the total silicon component content of the bioactivating substance calculated as silicon may be 1:3 to 1:10. The total silicon component content of the bioactivating substance includes silicon components naturally or originally present in the extract from activated animal tissues and any silicon components contributed by admixing the extract with additional silicon components not naturally or originally present in the extract from activated animal tissues.
The bioactivating substance may be obtained by activating or stressing animal tissue, grinding the activated animal tissues, adding a solvent for extraction to the ground tissue, removing the tissue pieces, removing protein from the remaining mixture, adsorbing the residue with an adsorbent, eluting the adsorbed components from the adsorbent, and then adding a predetermined amount of a soluble silicate to the resulting physiologically active substance as extracted above. The bioactivating substance of the present invention can also be obtained by a special extracting method,.i.e., all eluting operation from the adsorbent is carried out to make the content of silicic acid high.
In embodiments of the invention, extracts from an activated tissue which may be employed in the compositions and methods of the present invention may be extracts from inflammatory tissue inoculated with vaccinia virus disclosed in Japanese Examined Patent Publications Sho-631039,572 B, Sho-63/025,600 B and Hei-03/043,279 B, Japanese Patent No. 2,594,222 and U.S. Pat. No. 5,013,558 to Konishi and 5,560,935 to Konishi, et al. The disclosures of Japanese Examined Patent Publications Sho-63/039,572 B, Sho-63/025,600 B and Hei-03/043,279 B, Japanese Patent No. 2,594,222, and U.S. Pat. No. 5,013,558 to Konishi and U.S. Pat. No. 5,560,935 to Konishi, et al. are herein incorporated by reference in their entireties. A method for producing an extract from inflammatory tissue inoculated with vaccinia virus for use in the present invention is described, for example, in Example 1 of Japanese Examined Patent Publication Sho-63/039,572 B and Japanese Patent No. 2,594,222. Methods for producing extracts from inflammatory tissue inoculated with vaccinia virus for use in the present invention are also described, for example, in U.S. Pat. No. 5,013,558 to Konishi at column 1 line 44 to column 3 line 22, and in Examples 1 and 2 at column 3 lines 33 to 62, U.S. Pat. No. 5,560,935 to Konishi, et al. at column 2 line 55 to column 3 line 14, and column 3 line 33 to column 4 line 64, and in Examples 1 and 2 at column 5 line 6 to column 6 line 8, which are herein incorporated by reference in their entireties.
A commercially available drug preparation of an extract from inflammatory rabbit skin inoculated with vaccinia virus is sold in Japan under the trade name Neurotropin by Nippon Zoki Pharmaceutical Co., Osaka, Japan. As mentioned at pages 1,927 and 1,928 of xe2x80x9cDrugs in Japan, Ethical Drugsxe2x80x9d (22nd edition, 1998-1999; edited by Japan Pharmaceutical Information Center; published by Yakugyo Jiho Co., Ltd.), this preparation is a drug containing non-proteinous active substances extracted and isolated from inflammatory rabbit skin inoculated with vaccinia virus. The preparation has been used for treatment of lower back pain, neck-shoulder-arm syndrome, periarthritis scapulohumeralis, arthrosis deformans, symptomatic neuralgia, post-herpetic neuralgia, pruritis due to dermatological diseases (such as eczema, dermatitis and urticaria), allergic rhinitis, and sequelae of subacute myelo-optico-neuropathy (such as coldness, pain and paresthesia/dysesthesia). It is available as an ethical drug in the form of injections (subcutaneous, intramuscular and intravenous) and tablets.
Neurotropin was used in an experimental study at the School of Medicine, University of California, Davis, to evaluate its influence on thymic microenvironmental abnormalities of New England black mice as reported by Y. Takeoka et al, Int. J Immunotherapy, XI(2), pp. 49-56 (1995). As taught by Takeoka et al, Neurotropin is a non-protein extract isolated from the inflamed dermis of rabbits inoculated with vaccinia virus and it has been reported in the literature as: 1) having beneficial effects on immune-depressed animals, 2) clinically useful as an analgesic and as an anti-allergy drug with few side-effects in humans, 3) improving the immune status of murine lupus in (NZB/NZW) F1 mice, and 4) inhibiting the development of EAE in Lewis rats, an autoimmune model of human multiple sclerosis.
The commercially available extract, Neurotropin, may be used in the compositions and methods of the present invention. The descriptions, properties and dosages of Neurotropin reported in the above-mentioned xe2x80x9cDrugs in Japan, Ethical Drugsxe2x80x9d and the Takeoka et al article are incorporated herein by reference in their entireties.
The animal tissues used in the present invention are cultured tissues, cultured cells or inflammatory tissues of human or animal origin which are infected with virus, or chorio-allantoic membranes of embryonated eggs infected with virus. Examples of the virus used for the activation of the animal tissues as a stressor are orthopox viruses such as vaccinia virus, cowpox virus, variola virus, ectromelia virus and simian pox virus, parapoxviruses such as Orf virus, paravaccinia virus and bovine nopplelike stomatitis virus, goatpox viruses such as sheep pox virus, goatpox virus and lumpy skin disease virus, avian pox viruses such as avian pox virus and hare fibroma virus, rabbit pox viruses such as rabbit myxoma virus and rabbit fibroma virus, swine pox virus, Yava monkey tumor virus, Tara pox virus and other viruses belonging to the poxvirus family. As to tumor cells which may be used as a stressor, various tumor-cultured cell strains derived from human beings and animals may be used and anything that can be inoculated into the above animals and animal tissues to cause stress or activation can be employed. Mixtures of different strains of tumor cells can be inoculated into the animals and animal tissues to cause stress or activation. Also, the stressors may include combinations of viruses and tumor cells.
Exemplary of animals which may be used for preparing the activated tissues are domestic animals and fowl such as rabbits, cows, horses, sheep, goat, swine and chickens, and mammals such as monkeys, rats, mice, guinea pigs and hamsters. They may be selected depending upon the type of the stressors and the object or pharmaceutical effect desired. Regarding the cultured cells, any cell will do provided the stressor used is able to grow there. Examples of such cells which may be utilized for the culture are various tissues (e.g. human hemocytes and placentae) and the cultured cells of various tissues such as kidney, skin, lung, testis, lung, muscle, adrenal gland, thyroid gland, brain, nerve cells and hemocytes of the above-mentioned animals and embryos thereof.
The activated tissues or cells are aseptically collected, ground and made into an emulsified suspension by adding 1 to 5 times as much extracting solvent thereto. Examples of the extracting solvent applicable are distilled water, physiologically saline solution, weakly acidic or weakly basic buffers, etc. If necessary, stabilizers such as glycerol, antibacterial/antiseptic agents such as phenol, inorganic salts such as sodium chloride, potassium chloride and magnesium chloride may be added thereto in conventional amounts. At that time, the extraction can be facilitated by subjecting the admixture to treatment by means of freezing/melting, ultrasonic wave, cell membrane dissolving enzymes or surface-active agents.
The resulting milky extract is filtered or centrifuged to remove the tissue residue and then proteins are removed therefrom. Removal of the proteins can be carried out by known methods and treatments such as heating, ultrasonic waves, protein denaturing agents such as acids, bases, urea, guanidine, organic solvents and surface-active agents, isoelectric precipitation, salting-out, and the like. Then the proteins separated out therefrom are filtered off by means of filtration using filter paper (cellulose, nitrocellulose, etc.), a glass filter, Celite or a Seitz filter as well as ultrafiltration, gel filtration, an ion exchange resin, centrifugation and the like.
The resulting extracted fraction is adjusted to an acidic pH, preferably to pH 3.5-5.5, by an acid such as hydrochloric acid, sulfuric acid or hydrobromic acid, and adsorbed with an adsorbent. Examples of adsorbents which may be used are activated charcoal, kaolin and ion exchange resins. The adsorbent may be added to the extract followed by stirring or the extract may be passed through a column filled with the adsorbent whereby the effective component can be adsorbed.
In eluting the physiologically active substance of the present invention from the adsorbent, an extracting solvent (e.g. a basic aqueous solution, a solution in a water-miscible solvent such as alcohol or a mixed solution thereof) may be added. The mixture is preferably adjusted to pH 9-12, and then the physiologically active substance is eluted at room temperature or by heating appropriately or with stirring. The absorbent is removed by a conventional manner such as filtration to complete the elution. Then, if necessary, means such as chromatography, ultrafiltration and dialysis using a reverse osmosis filtration or removal of the salt therefrom may be applied whereupon the physiologically active substance can be prepared in a more purified state. The resulting physiologically active substance extracted from the activated animal tissues as above contains silicon components in an amount of 1-20 xcexcg which are calculated as silicon per mg of dried substance.
The silicon substances which are added to the above physiologically active substance to prepare the bioactivating substance of the present invention are water-soluble silicic acids, water-soluble silicates, polymers of water-soluble silicic acids, polymers of water-soluble silicates, or mixtures thereof. For example, the silicon components may each be added in its own form or a polymerized form of silicic acid such as orthosilicic acid, metasilicic acid, mesodisilicic acid, mesotrisilicic acid and mesotetrasilicic acid and salts thereof with an alkali or alkaline metal such as sodium and potassium or water glass. It is also possible to add light and heavy anhydrous silicic acid in a fused state in an alkaline solution. A substance prepared by an alkali fusion of minerals containing a high content of silicon such as crystal, quartz, feldspar, granite and periodotite can be added. A substance prepared by treating diatomaceous earth, bentonite glass or active carbon with an alkaline substance can also be employed. It is possible to add a soluble silicate extracted from a plant, animal or diatomaceous earth containing a high content of silicon, for example, scouring rush, murasaki reishi (a kind of bracket fungus of the genus Fomes), tochu (bark of Eucommia ulmoides), rice plant, barley, bamboo, susuki (Japanese pampas grass), field horsetail, sponge and diatom.
Silicate polymers which may be employed in the present invention for admixture with the extracts of the present invention may be silicate polymers disclosed in U.S. Pat. No. 5,534,509 to Konishi et al. The disclosure of U.S. Pat. No.5,534,509 is herein incorporated by reference in its entirety.
It is further possible that, in the manufacture of the physiologically active substance from the activated tissues eluted from an adsorbent such as active carbon, kaolin and bentonite, the pH is raised, the elution time is extended or the elution temperature is raised. According to the elution operation, extra silicon substances from the adsorbent are mixed therewith to prepare the bioactivating substance of the present invention containing a high amount of silicic acid.
The silicon compound may be appropriately added to the extracted substance from the activated tissues depending upon the object for use or within such an extent that the function is not deteriorated. The silicon components contained in the substance of the present invention are water-soluble silicic acid or its polymer where silicates are polymerized. They may be present in a single or polymerized form of silicic acid such as orthosilicic acid, metasilicic acid, mesodisilicic acid, mesotrisilicic acid and mesotetrasilicic acid and salts thereof with an alkali or alkaline metal such as sodium and potassium. The bioactivating substance of the present invention contains such silicon substances in an amount of more than 20 xcexcg, for example more than 22 xcexcg, preferably more than 25 xcexcg, which are calculated as silicon per mg of dried substance.
Preferred embodiments of the present invention are:
(1) A bioactivating substance which is extracted and manufactured from activated tissues, shows positive color reactions to amino acid (by a ninhydrin reaction), saccharide (by an orcinol-iron (III) chloride-hydrochloric acid method), phosphorus (by a molybdenum blue method) and silicic acid (by a molybdenum blue method), shows negative qualitative reactions to protein (by a trichloroacetic acid method) and phenol (by a ferric chloride method) and contains silicon components in an amount of more than 20 xcexcg which are calculated as silicon per mg of dried substance.
(2) The bioactivating substance according to the above paragraph (1), wherein silicate is added to the substance extracted from the activated tissues.
(3) The bioactivating substance according to the above paragraph (1), wherein silicate polymer is added to the substance extracted from the activated tissues.
(4) The bioactivating substance according to the above paragraph (1), wherein silicate prepared by dissolving silicon oxide with sodium hydroxide or potassium hydroxide is added to the substance extracted from the activated tissues.
(5) The bioactivating substance according to the above paragraph (1), wherein a soluble silicate extracted from plant, animal or diatomaceous earth containing a high content of silicon such as scouring rush, murasaki reishi (a kind of bracket fungus of the genus Fomes), tochu (bark of Eucommia ulmoides), rice plant, barley, bamboo, susuki (Japanese pampas grass), field horsetail, sponge and diatom is added to the substance extracted from the activated tissues.
(6) The bioactivating substance according to the above paragraph (1), wherein silicic acid prepared by the extraction of minerals containing a high content of silicon (such as crystal, quartz, feldspar, granite, periodotite and glass) with an alkali is added to the substance extracted from the activated tissues.
(7) The bioactivating substance according to the above paragraph (1), wherein the said bioactivating substance is obtained by increasing the addition of silicon in the eluting operation from the adsorbent during the extraction from the activated tissues.
The following non-limiting examples illustrate methods for the manufacture of the product of the present invention and physiological effectiveness of the product wherein all parts, percentages and ratios are by weight, all temperatures are in xc2x0 C., and all reactions are conducted at about atmospheric pressure unless indicated to the contrary: